{"gene":"IGSF8","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2001,"finding":"EWI-2 (IGSF8) was co-purified with tetraspanin CD81 under stringent detergent conditions and forms highly stoichiometric, direct complexes with both CD9 and CD81 (but not other tetraspanins or integrins), as shown by co-purification, immunodepletion, and covalent cross-linking experiments.","method":"Co-purification, mass spectrometry, immunodepletion, chemical cross-linking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (co-purification, cross-linking, immunodepletion) in a highly-cited foundational paper","pmids":["11504738"],"is_preprint":false},{"year":2001,"finding":"The interaction of CD81 with PGRL (EWI-2/IGSF8) requires the membrane-distal Ig3-Ig4 domains of PGRL, as determined by coexpression of domain-deletion constructs.","method":"Domain-deletion constructs, co-immunoprecipitation","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping with expression constructs, single lab","pmids":["11673522"],"is_preprint":false},{"year":2003,"finding":"Two separate regions of CD9 (of 40 and 47 amino acids) confer the ability to interact with EWI-2, as demonstrated using chimeric CD9/CD82 molecules.","method":"Chimeric CD9/CD82 constructs, co-immunoprecipitation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping with chimeric proteins, single lab","pmids":["12708969"],"is_preprint":false},{"year":2003,"finding":"EWI-2 (IGSF8) physically associates with the metastasis suppressor KAI1/CD82 in a highly stoichiometric, direct interaction independent of cholesterol-enriched lipid rafts, and overexpression of EWI-2 in prostate cancer cells inhibits cell migration on fibronectin and laminin substrata.","method":"Chemical cross-linking, co-immunoprecipitation, cell migration assay (overexpression)","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — direct association established by cross-linking and co-IP, functional overexpression assay","pmids":["12750295"],"is_preprint":false},{"year":2003,"finding":"EWI-2 overexpression impairs alpha3beta1 integrin-dependent cell reaggregation and motility on laminin-5; tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin (but not other integrins), and CD81 controls EWI-2 maturation and cell surface localization.","method":"Overexpression, immunodepletion, re-expression, cell migration/reaggregation assay, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including immunodepletion, re-expression, and chimeric mutant controls in a well-cited study","pmids":["14662754"],"is_preprint":false},{"year":2006,"finding":"EWI-2 directly associates with ezrin-radixin-moesin (ERM) proteins via a basic charged stretch in its cytoplasmic domain, co-localizes with ERMs at microspikes and cellular uropods, and silencing of EWI-2 augments cell migration, polarity, and ERM phosphorylation.","method":"GST pulldown, protein-protein binding assay, co-immunoprecipitation, confocal microscopy, dominant-negative moesin, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — direct in vitro binding assay (GST fusion) combined with dominant-negative experiment and siRNA functional readout, strongly replicated across multiple approaches","pmids":["16690612"],"is_preprint":false},{"year":2003,"finding":"EWI-2 modulates alpha4beta1 integrin function: wild-type EWI-2 overexpression impairs lymphocyte spreading and ruffling on VCAM-1, reorganizes cell-surface CD81, and increases alpha4beta1-CD81 complex size; a cytoplasmic-tail mutant EWI-2 lacks these effects and fails to associate with alpha4beta1 and CD81.","method":"Co-immunoprecipitation, size exclusion chromatography, shear flow adhesion assay, overexpression/mutant analysis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, mutant controls, and functional assays in the same study","pmids":["15070678"],"is_preprint":false},{"year":2007,"finding":"EWI-2/CD316 functions as a receptor for heat shock protein HSPA8 on dendritic cells; soluble EWI-2 binds both HSPA8-expressing cells and immobilized HSPA8 protein, and EWI-2 ligation enhances CCL21-dependent migration of mature dendritic cells while attenuating their antigen-specific stimulatory capacity.","method":"Expression cloning, binding assay (soluble EWI-2 to cells/immobilized protein), functional migration and T cell stimulation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — expression cloning identification of ligand, direct binding assay, functional assays in a single study","pmids":["17785435"],"is_preprint":false},{"year":2009,"finding":"EWI-2 loss from the oocyte surface in CD9-null mice (reduced to <10% of wild-type levels) depends on CD9 expression, indicating CD9 is required for normal surface localization of EWI-2 on eggs.","method":"Genetic knockout (CD9-null mice), flow cytometry/surface protein quantification","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic KO with direct protein quantification, single lab","pmids":["19107828"],"is_preprint":false},{"year":2009,"finding":"EWI-2 participates in mouse sperm-egg interaction: anti-IgSF8 antibody has moderate inhibitory effects on sperm-egg binding, while CD9 perturbation significantly inhibits fusion, suggesting IgSF8 and CD9 play discrete roles in gamete interaction.","method":"Antibody-mediated perturbation, in vitro fertilization assay","journal":"Reproduction, fertility, and development","confidence":"Medium","confidence_rationale":"Tier 2 — functional antibody perturbation assay with direct comparison to CD9, single lab","pmids":["19210920"],"is_preprint":false},{"year":2009,"finding":"EWI-2 expression in glioblastoma cells reorganizes tetraspanins CD9 and CD81 (increasing their mutual association) and dissociates CD81 and other tetraspanins from MMP-2 and MT1-MMP, thereby inhibiting invasion and colony formation.","method":"Overexpression, co-immunoprecipitation, soft agar and invasion assays, in vivo tumor xenograft","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP showing molecular reorganization combined with in vitro and in vivo functional assays, single lab","pmids":["19107234"],"is_preprint":false},{"year":2011,"finding":"A glycine zipper motif in the transmembrane domain of EWI-2 is essential for its interaction with CD81, and palmitoylation on two juxtamembranous cytosolic cysteines is required for EWI-2 interaction with both CD81 and CD9; PIP binding regulates palmitoylation and protein stability but not vice versa.","method":"Mutagenesis, palmitoylation assay, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — active-site/domain mutagenesis with direct biochemical readouts for both transmembrane glycine zipper and palmitoylation sites","pmids":["21343309"],"is_preprint":false},{"year":2011,"finding":"EWI2 cytoplasmic tail directly binds phosphatidylinositol phosphates (PIPs) via two basic residue clusters; palmitoylation at N-terminal cytoplasmic cysteines is required for tetraspanin-EWI2 association and EWI2-dependent inhibition of cell migration and lamellipodia formation; the two modifications have differential and opposing roles in cell-cell adhesion and proliferation.","method":"Lipid-binding assay, site-directed mutagenesis, palmitoylation assay, co-immunoprecipitation, cell migration/lamellipodia assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro lipid-binding assay, mutagenesis of both interaction sites, functional readouts for migration and adhesion","pmids":["21609323"],"is_preprint":false},{"year":2012,"finding":"EWI-2 colocalizes with CD3 and CD81 at the central SMAC of the T cell immune synapse; mass spectrometry of EWI-2 cytoplasmic domain pulldowns identified α-actinin as a direct binding partner, regulated by PIP2; silencing of EWI-2 or a cytoplasmic truncation mutant increases IL-2 secretion upon antigen stimulation; EWI-2 and α-actinin-4 silencing each increase HIV cell infectivity.","method":"Co-immunoprecipitation, mass spectrometry pulldown, confocal microscopy, siRNA knockdown, cell-cell fusion assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 — MS identification of binding partner confirmed by co-IP, PIP2 regulation shown biochemically, functional assays with siRNA and dominant-negative mutant","pmids":["22689882"],"is_preprint":false},{"year":2012,"finding":"IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (shown by immunoprecipitation) and localizes to puncta within axons and growth cones of olfactory sensory neurons consistent with tetraspanin-enriched microdomain localization; IgSF8 glomerular expression is transitory during synapse formation and is re-induced upon OSN regeneration.","method":"Co-immunoprecipitation, immunofluorescence/confocal microscopy, lesion/regeneration model","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP plus localization during synaptogenesis, single lab, no direct functional manipulation","pmids":["22687584"],"is_preprint":false},{"year":2015,"finding":"EWI-2 negatively regulates TGF-β signaling in melanoma by sequestering CD9 and CD81 away from TGF-β receptors; when EWI-2 is knocked down, CD9 and CD81 become available to support TβR2-TβR1 association, markedly elevating TGF-β signaling and downstream EMT, migration, invasion, and metastasis.","method":"RNAi, overexpression, co-immunoprecipitation, in vitro cytostasis/migration/invasion assays, in vivo lung metastasis model","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 — epistasis established by showing CD9/CD81 depletion reverses EWI-2 knockdown phenotypes; multiple orthogonal assays in vitro and in vivo","pmids":["25656846"],"is_preprint":false},{"year":2019,"finding":"EWI-2 accumulates at the presynaptic terminal of the HIV virological synapse and inhibits Env-mediated cell-cell fusion; EWI-2 is downregulated upon HIV-1 infection (likely by Vpu); EWI-2 and CD81 levels are restored on syncytia surfaces, contributing to prevention of further fusion.","method":"Quantitative fluorescence microscopy, shRNA knockdown, cell-cell fusion assay","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by microscopy and functional siRNA knockdown with fusion readout, single lab","pmids":["31757023"],"is_preprint":false},{"year":2020,"finding":"IgSF8 is a neuronal receptor enriched in the hippocampal mossy fiber (MF) pathway; presynaptic Igsf8 deletion impairs MF synaptic architecture, robustly decreases bouton filopodia density (providing feedforward inhibition), disrupts excitation/inhibition balance, and increases CA3 pyramidal neuron excitability; cell-surface interactome screening identified IgSF8 binding partners at MF synapses.","method":"Cell-surface interactome screening, presynaptic conditional knockout, electron microscopy, electrophysiology, synaptome proteomics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — conditional KO with multiple readouts (ultrastructure, electrophysiology, proteomics), interactome screening","pmids":["33057002"],"is_preprint":false},{"year":2021,"finding":"EWI-2 localizes not only on the plasma membrane but also on the nuclear envelope, where it regulates nuclear translocation of EGFR signaling molecules and sorting of miR-3934-5p between cells and exosomes, acting through the EGFR-MAPK-ERK pathway to inhibit prostate cancer cell metastasis.","method":"Subcellular fractionation/localization, overexpression/knockdown, miRNA quantification, signaling pathway analysis","journal":"Molecular oncology","confidence":"Low","confidence_rationale":"Tier 3 — novel nuclear envelope localization claim with limited mechanistic validation; single lab, partial follow-up","pmids":["33605506"],"is_preprint":false},{"year":2022,"finding":"EWI2 prevents EGFR clustering, endocytosis, and activation; upon EWI2 silencing or ablation, EGFR clustering and endocytosis increase, leading to elevated ERK MAP kinase signaling, partial EMT, and increased cancer cell proliferation and metastatic potential; EGFR or ERK inhibition abrogates these phenotypes.","method":"siRNA/gene ablation, single-molecule localization microscopy (EGFR clustering), endocytosis assay, signaling pathway inhibition, in vivo xenograft","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 1-2 — super-resolution imaging of EGFR clustering, rescue by pathway inhibitors, in vivo validation, multiple orthogonal methods","pmids":["35773608"],"is_preprint":false},{"year":2022,"finding":"EWI2 promotes endolysosomal degradation of growth factor receptors and integrins by elevating lysosome formation via nuclear retention of TFEB (the master lysosomogenesis transcription factor), reducing cell-surface levels of these receptors and suppressing lung cancer growth and metastasis.","method":"EWI2 forced expression, lysosome assays, receptor surface/total level quantification, TFEB nuclear localization assay, in vivo tumor model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 — TFEB nuclear retention mechanistically linked to lysosome formation and receptor degradation with in vivo validation, single lab","pmids":["35339615"],"is_preprint":false},{"year":2024,"finding":"IGSF8 expressed on tumor cells suppresses NK cell function by directly interacting with inhibitory NK cell receptors KIR3DL2 (human) and Klra9 (mouse); an antibody blocking this interaction enhances NK cell killing in vitro and upregulates antigen presentation, NK cytotoxicity, and T cell signaling in vivo, inhibiting tumor growth alone or in combination with anti-PD1.","method":"CRISPR screen, co-immunoprecipitation/interaction assay, antibody blockade, in vitro NK killing assay, syngeneic tumor models","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 — CRISPR screen discovery validated by direct binding, functional in vitro and in vivo assays, antibody rescue experiment with multiple orthogonal readouts","pmids":["38657602"],"is_preprint":false}],"current_model":"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 (requiring a glycine zipper in its transmembrane domain and palmitoylation of cytoplasmic cysteines), links tetraspanin-enriched microdomains to the actin cytoskeleton via direct binding to ERM proteins and α-actinin (regulated by PIP2 and PIPs), modulates integrin (α3β1, α4β1) and growth factor receptor (EGFR) function by controlling their clustering, endocytosis, and endolysosomal degradation, negatively regulates TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, acts as an innate immune checkpoint by interacting with NK cell inhibitory receptors KIR3DL2/Klra9, organizes hippocampal CA3 microcircuit connectivity as a presynaptic regulator of mossy fiber bouton filopodia and E/I balance, and inhibits HIV-induced cell-cell fusion at the virological presynapse."},"narrative":{"teleology":[{"year":2001,"claim":"Identification of IGSF8 as a direct, highly stoichiometric partner of tetraspanins CD9 and CD81 established it as a core component of tetraspanin-enriched microdomains, resolving which IgSF proteins are bona fide tetraspanin partners versus fortuitous co-precipitants.","evidence":"Co-purification, mass spectrometry, chemical cross-linking, and immunodepletion in multiple cell lines; domain-deletion mapping of the Ig3–Ig4 domains required for CD81 binding","pmids":["11504738","11673522"],"confidence":"High","gaps":["Structural basis of the IGSF8–tetraspanin interface not resolved at atomic level","Whether IGSF8 can engage CD9 and CD81 simultaneously or competitively was not determined"]},{"year":2003,"claim":"IGSF8 was shown to modulate integrin-dependent adhesion and migration by reorganizing tetraspanin–integrin complexes, revealing a mechanism by which IgSF8 indirectly controls integrin function through tetraspanin scaffolding rather than direct integrin binding.","evidence":"Overexpression and cytoplasmic-tail mutant analysis in cell reaggregation, migration, and shear-flow adhesion assays; co-IP with α3β1 and α4β1 integrins; chimeric CD9/CD82 domain mapping of the EWI-2 binding regions on CD9","pmids":["14662754","15070678","12708969","12750295"],"confidence":"High","gaps":["Whether IGSF8 controls integrin signaling through conformational or purely spatial mechanisms was not distinguished","Endogenous stoichiometry of IGSF8–tetraspanin–integrin complexes in primary cells not established"]},{"year":2006,"claim":"Discovery that IGSF8 directly binds ERM proteins via a basic cytoplasmic stretch and that its silencing augments ERM phosphorylation and cell migration established IGSF8 as a negative regulator linking tetraspanin microdomains to the cortical actin cytoskeleton.","evidence":"GST pulldown (direct binding), confocal co-localization at microspikes/uropods, dominant-negative moesin, siRNA knockdown","pmids":["16690612"],"confidence":"High","gaps":["Whether ERM binding and tetraspanin binding are mutually exclusive or cooperative was not resolved","Upstream signals controlling IGSF8–ERM dissociation remain undefined"]},{"year":2011,"claim":"Mutagenesis of the transmembrane glycine zipper and cytoplasmic palmitoylation sites, together with phosphoinositide-binding mapping, revealed the post-translational logic governing IGSF8 assembly into tetraspanin webs and its regulation of migration and adhesion.","evidence":"Site-directed mutagenesis of TM glycine zipper and palmitoylation cysteines, in vitro lipid-binding assays, palmitoylation assays, functional migration/lamellipodia assays","pmids":["21343309","21609323"],"confidence":"High","gaps":["Identity of the palmitoyl acyltransferase(s) modifying IGSF8 unknown","How PIP binding and palmitoylation are dynamically coordinated in live cells not shown"]},{"year":2012,"claim":"Identification of α-actinin as a PIP2-regulated direct cytoplasmic partner of IGSF8, and the finding that IGSF8 localizes to the immune synapse cSMAC where its loss elevates IL-2 secretion and HIV infectivity, established IGSF8 as a functional brake on T cell activation and viral entry.","evidence":"Mass spectrometry pulldown, co-IP, PIP2 regulation, confocal at immune synapse, siRNA/cytoplasmic-truncation functional assays, cell–cell fusion assay","pmids":["22689882"],"confidence":"High","gaps":["Whether α-actinin and ERM binding are competitive or spatially segregated was not tested","Mechanism by which IGSF8 restrains IL-2 signaling beyond clustering is undefined"]},{"year":2015,"claim":"Epistasis experiments demonstrated that IGSF8 negatively regulates TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, so that IGSF8 loss frees tetraspanins to promote TβR2–TβR1 association, driving EMT and metastasis — a new signaling-level mechanism for IGSF8's tumor-suppressive activity.","evidence":"RNAi/overexpression epistasis (CD9/CD81 depletion rescues EWI-2 knockdown phenotype), co-IP of TGF-β receptor complexes, in vivo lung metastasis model","pmids":["25656846"],"confidence":"High","gaps":["Whether IGSF8 physically contacts TGF-β receptors or acts exclusively through tetraspanin redistribution not clarified","Applicability beyond melanoma not tested"]},{"year":2020,"claim":"Conditional presynaptic knockout of Igsf8 in hippocampal mossy fibers revealed a non-immune, neurodevelopmental role: IGSF8 is required for bouton filopodia formation and excitation/inhibition balance in CA3 circuits, establishing it as a synaptic organizer.","evidence":"Presynaptic conditional knockout, electron microscopy, electrophysiology, cell-surface interactome screening","pmids":["33057002"],"confidence":"High","gaps":["Trans-synaptic binding partners mediating filopodia formation not fully identified","Whether IGSF8 synaptic function depends on tetraspanin association was not tested"]},{"year":2022,"claim":"Super-resolution imaging and gene ablation showed that IGSF8 prevents EGFR clustering and endocytosis, and that IGSF8 promotes endolysosomal degradation of growth factor receptors and integrins via TFEB-dependent lysosomogenesis, providing two complementary mechanisms for receptor downregulation and tumor suppression.","evidence":"Single-molecule localization microscopy (EGFR clustering), endocytosis assays, TFEB nuclear localization assay, EGFR/ERK inhibitor rescue, in vivo xenograft and lung cancer models","pmids":["35773608","35339615"],"confidence":"High","gaps":["How IGSF8 promotes TFEB nuclear retention mechanistically is unclear","Whether EGFR clustering prevention and lysosome-mediated degradation operate in the same or distinct cell contexts is unresolved"]},{"year":2024,"claim":"CRISPR screening and direct binding studies revealed IGSF8 as an innate immune checkpoint: tumor-expressed IGSF8 engages inhibitory NK receptors KIR3DL2/Klra9 to suppress NK cell killing, and antibody blockade of this axis enhances antitumor immunity alone or with anti-PD1.","evidence":"CRISPR screen, co-immunoprecipitation/interaction assay, antibody blockade, in vitro NK killing assay, syngeneic tumor models","pmids":["38657602"],"confidence":"High","gaps":["Structural basis of IGSF8–KIR3DL2 interaction not determined","Whether tetraspanin association modulates the immune checkpoint function is unknown","Patient-level correlation between IGSF8 expression and NK cell infiltration/response to immunotherapy not yet established"]},{"year":null,"claim":"Key unresolved questions include the structural basis of IGSF8's multivalent interactions (tetraspanins, KIR3DL2, ERM, α-actinin), whether its immune checkpoint and tetraspanin-scaffolding functions are mechanistically coupled, and the identity of its trans-synaptic partners in mossy fiber circuits.","evidence":"","pmids":[],"confidence":"Low","gaps":["No atomic-resolution structure of IGSF8 or its complexes available","Functional interdependence of immune checkpoint and tetraspanin-organizing roles untested","Trans-synaptic ligands at mossy fiber synapses remain unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,6,15,19,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[5,13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,11,15]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,5,8,13,16,17,21]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[15,19,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,21]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[4,6,9]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[10,15,19,20]}],"complexes":["Tetraspanin-enriched microdomain (CD9/CD81-EWI-2)"],"partners":["CD9","CD81","EZR","ACTN4","KIR3DL2","CD82","EGFR","HSPA8"],"other_free_text":[]},"mechanistic_narrative":"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that organizes tetraspanin-enriched microdomains and modulates integrin, growth factor receptor, and immune receptor signaling. It forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 — requiring a transmembrane glycine zipper motif and palmitoylation of cytoplasmic cysteines — and links these microdomains to the actin cytoskeleton through direct binding of ERM proteins and α-actinin, regulated by phosphoinositide lipids [PMID:11504738, PMID:21343309, PMID:16690612, PMID:22689882]. IGSF8 negatively regulates cell migration, EGFR clustering/endocytosis, and TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors and by promoting endolysosomal degradation of growth factor receptors and integrins via TFEB-dependent lysosomogenesis, thereby suppressing EMT and metastasis [PMID:25656846, PMID:35773608, PMID:35339615]. IGSF8 also functions as an innate immune checkpoint that suppresses NK cell cytotoxicity through direct interaction with inhibitory receptors KIR3DL2 and Klra9, and serves as a presynaptic regulator in hippocampal mossy fiber circuits controlling bouton filopodia density and excitation/inhibition balance [PMID:38657602, PMID:33057002]."},"prefetch_data":{"uniprot":{"accession":"Q969P0","full_name":"Immunoglobulin superfamily member 8","aliases":["CD81 partner 3","Glu-Trp-Ile EWI motif-containing protein 2","EWI-2","Keratinocytes-associated transmembrane protein 4","KCT-4","LIR-D1","Prostaglandin regulatory-like protein","PGRL"],"length_aa":613,"mass_kda":65.0,"function":"Member of the immunoglobulin superfamily (IgSF) that links tetraspanin-enriched microdomains to the actin cytoskeleton and plays several important roles in innate and adaptive immunity (PubMed:11504738, PubMed:14662754). Acts as an inducible receptor of HSPA8 on dendritic cells to enhance the CCL21/SLC-dependent migration of activated mature dendritic cells while attenuating their antigen-specific stimulatory capacities (PubMed:17785435). In complex with alpha-actinins ACTN1 and ACTN4, regulates actin dynamics in the immune synapse and subsequent T-cell activation (PubMed:22689882). Inhibits the entry of several viruses such as hepatitis C Virus (HCV) or HIV-1. Mechanistically, promotes a change in CD81 organization at the plasma membrane by significantly restricting its diffusion which in turn influences CD81 interaction with Claudin-1/CLDN1, preventing CLDN1 from acting as a co-receptor required for HCV entry (PubMed:23351194). Accumulates at the presynaptic terminal, the producer cell side of the virological synapse, to prevent HIV-1 Env-mediated cell-cell fusion (PubMed:31757023). Highly expressed on malignant cells with antigen presentation defects, interacts with NK receptor KIR3DL2 to suppress NK-cell cytotoxicity (PubMed:38657602). May participate in the regulation of neurite outgrowth and maintenance of the neural network in the adult brain","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q969P0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IGSF8","classification":"Not Classified","n_dependent_lines":14,"n_total_lines":1208,"dependency_fraction":0.011589403973509934},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IGSF8","total_profiled":1310},"omim":[{"mim_id":"606644","title":"IMMUNOGLOBULIN SUPERFAMILY, MEMBER 8; IGSF8","url":"https://www.omim.org/entry/606644"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":153.9}],"url":"https://www.proteinatlas.org/search/IGSF8"},"hgnc":{"alias_symbol":["CD81P3","EWI2","PGRL","CD316"],"prev_symbol":[]},"alphafold":{"accession":"Q969P0","domains":[{"cath_id":"2.60.40.10","chopping":"28-148","consensus_level":"high","plddt":95.1681,"start":28,"end":148},{"cath_id":"2.60.40.10","chopping":"152-160_171-298","consensus_level":"high","plddt":93.3626,"start":152,"end":298},{"cath_id":"2.60.40.10","chopping":"305-433","consensus_level":"high","plddt":90.9349,"start":305,"end":433},{"cath_id":"2.60.40.10","chopping":"439-483_494-572","consensus_level":"high","plddt":89.969,"start":439,"end":572}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969P0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q969P0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q969P0-F1-predicted_aligned_error_v6.png","plddt_mean":88.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IGSF8","jax_strain_url":"https://www.jax.org/strain/search?query=IGSF8"},"sequence":{"accession":"Q969P0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q969P0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q969P0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969P0"}},"corpus_meta":[{"pmid":"11504738","id":"PMC_11504738","title":"EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11504738","citation_count":190,"is_preprint":false},{"pmid":"16690612","id":"PMC_16690612","title":"EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16690612","citation_count":165,"is_preprint":false},{"pmid":"12708969","id":"PMC_12708969","title":"EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.","date":"2003","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12708969","citation_count":117,"is_preprint":false},{"pmid":"12750295","id":"PMC_12750295","title":"EWI2/PGRL associates with the metastasis suppressor KAI1/CD82 and inhibits the migration of prostate cancer cells.","date":"2003","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/12750295","citation_count":88,"is_preprint":false},{"pmid":"11673522","id":"PMC_11673522","title":"PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.","date":"2001","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/11673522","citation_count":74,"is_preprint":false},{"pmid":"14662754","id":"PMC_14662754","title":"EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5.","date":"2003","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/14662754","citation_count":72,"is_preprint":false},{"pmid":"15070678","id":"PMC_15070678","title":"EWI-2 modulates lymphocyte integrin alpha4beta1 functions.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15070678","citation_count":51,"is_preprint":false},{"pmid":"21343309","id":"PMC_21343309","title":"Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21343309","citation_count":50,"is_preprint":false},{"pmid":"25656846","id":"PMC_25656846","title":"EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis.","date":"2015","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/25656846","citation_count":48,"is_preprint":false},{"pmid":"33057002","id":"PMC_33057002","title":"Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33057002","citation_count":44,"is_preprint":false},{"pmid":"22689882","id":"PMC_22689882","title":"EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection.","date":"2012","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/22689882","citation_count":43,"is_preprint":false},{"pmid":"19107234","id":"PMC_19107234","title":"Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo.","date":"2009","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/19107234","citation_count":42,"is_preprint":false},{"pmid":"38657602","id":"PMC_38657602","title":"IGSF8 is an innate immune checkpoint and cancer immunotherapy target.","date":"2024","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/38657602","citation_count":40,"is_preprint":false},{"pmid":"19210920","id":"PMC_19210920","title":"Immunoglobulin superfamily member IgSF8 (EWI-2) and CD9 in fertilisation: evidence of distinct functions for CD9 and a CD9-associated protein in mammalian sperm-egg interaction.","date":"2009","source":"Reproduction, fertility, and development","url":"https://pubmed.ncbi.nlm.nih.gov/19210920","citation_count":32,"is_preprint":false},{"pmid":"33605506","id":"PMC_33605506","title":"EWI-2 controls nucleocytoplasmic shuttling of EGFR signaling molecules and miRNA sorting in exosomes to inhibit prostate cancer cell metastasis.","date":"2021","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33605506","citation_count":21,"is_preprint":false},{"pmid":"17785435","id":"PMC_17785435","title":"EWI-2/CD316 is an inducible receptor of HSPA8 on human dendritic cells.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17785435","citation_count":20,"is_preprint":false},{"pmid":"22609062","id":"PMC_22609062","title":"Tetraspanin-interacting protein IGSF8 is dispensable for mouse fertility.","date":"2012","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/22609062","citation_count":17,"is_preprint":false},{"pmid":"26989766","id":"PMC_26989766","title":"Novel impact of EWI-2, CD9, and CD81 on TGF-β signaling in melanoma.","date":"2015","source":"Molecular & cellular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/26989766","citation_count":16,"is_preprint":false},{"pmid":"21609323","id":"PMC_21609323","title":"Differential functions of phospholipid binding and palmitoylation of tumour suppressor EWI2/PGRL.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21609323","citation_count":15,"is_preprint":false},{"pmid":"22687584","id":"PMC_22687584","title":"IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses.","date":"2012","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/22687584","citation_count":15,"is_preprint":false},{"pmid":"31757023","id":"PMC_31757023","title":"EWI-2 Inhibits Cell-Cell Fusion at the HIV-1 Virological Presynapse.","date":"2019","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/31757023","citation_count":12,"is_preprint":false},{"pmid":"19107828","id":"PMC_19107828","title":"Loss of surface EWI-2 on CD9 null oocytes.","date":"2009","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/19107828","citation_count":12,"is_preprint":false},{"pmid":"36788350","id":"PMC_36788350","title":"EWI2 and its relatives in Tetraspanin-enriched membrane domains regulate malignancy.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36788350","citation_count":11,"is_preprint":false},{"pmid":"12595239","id":"PMC_12595239","title":"Genomic organization and embryonic expression of Igsf8, an immunoglobulin superfamily member implicated in development of the nervous system and organ epithelia.","date":"2003","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/12595239","citation_count":10,"is_preprint":false},{"pmid":"35773608","id":"PMC_35773608","title":"EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.","date":"2022","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/35773608","citation_count":8,"is_preprint":false},{"pmid":"35339615","id":"PMC_35339615","title":"EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.","date":"2022","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/35339615","citation_count":6,"is_preprint":false},{"pmid":"38453613","id":"PMC_38453613","title":"IGSF8 is a potential target for the treatment of gliomas.","date":"2024","source":"Asian journal of surgery","url":"https://pubmed.ncbi.nlm.nih.gov/38453613","citation_count":5,"is_preprint":false},{"pmid":"28544021","id":"PMC_28544021","title":"Differential expression of EWI-2 in endometriosis, its functional role and underlying molecular mechanisms.","date":"2017","source":"The journal of obstetrics and gynaecology research","url":"https://pubmed.ncbi.nlm.nih.gov/28544021","citation_count":4,"is_preprint":false},{"pmid":"39755227","id":"PMC_39755227","title":"IGSF8 impairs migration and invasion of trophoblast cells and angiogenesis in preeclampsia.","date":"2025","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/39755227","citation_count":1,"is_preprint":false},{"pmid":"1840028","id":"PMC_1840028","title":"Thymic origin of some natural killer cells: clonal proliferation of human CD3-16+ cells from CD3-4-8- thymocyte precursors requires the presence of H9 leukemic cells.","date":"1991","source":"International journal of clinical & laboratory research","url":"https://pubmed.ncbi.nlm.nih.gov/1840028","citation_count":1,"is_preprint":false},{"pmid":"40936932","id":"PMC_40936932","title":"Functional characterization and clinical significance of IGSF8 in pan-cancer: an integrated bioinformatic and experimental study.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40936932","citation_count":0,"is_preprint":false},{"pmid":"41346582","id":"PMC_41346582","title":"R3HDM4 influences kidney renal clear cell carcinoma progression, immune modulation, and potential links to the IGSF8 immune checkpoint.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41346582","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16910,"output_tokens":5146,"usd":0.06396},"stage2":{"model":"claude-opus-4-6","input_tokens":8727,"output_tokens":3266,"usd":0.187927},"total_usd":0.251887,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"EWI-2 (IGSF8) was co-purified with tetraspanin CD81 under stringent detergent conditions and forms highly stoichiometric, direct complexes with both CD9 and CD81 (but not other tetraspanins or integrins), as shown by co-purification, immunodepletion, and covalent cross-linking experiments.\",\n      \"method\": \"Co-purification, mass spectrometry, immunodepletion, chemical cross-linking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (co-purification, cross-linking, immunodepletion) in a highly-cited foundational paper\",\n      \"pmids\": [\"11504738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The interaction of CD81 with PGRL (EWI-2/IGSF8) requires the membrane-distal Ig3-Ig4 domains of PGRL, as determined by coexpression of domain-deletion constructs.\",\n      \"method\": \"Domain-deletion constructs, co-immunoprecipitation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with expression constructs, single lab\",\n      \"pmids\": [\"11673522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two separate regions of CD9 (of 40 and 47 amino acids) confer the ability to interact with EWI-2, as demonstrated using chimeric CD9/CD82 molecules.\",\n      \"method\": \"Chimeric CD9/CD82 constructs, co-immunoprecipitation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with chimeric proteins, single lab\",\n      \"pmids\": [\"12708969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2 (IGSF8) physically associates with the metastasis suppressor KAI1/CD82 in a highly stoichiometric, direct interaction independent of cholesterol-enriched lipid rafts, and overexpression of EWI-2 in prostate cancer cells inhibits cell migration on fibronectin and laminin substrata.\",\n      \"method\": \"Chemical cross-linking, co-immunoprecipitation, cell migration assay (overexpression)\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct association established by cross-linking and co-IP, functional overexpression assay\",\n      \"pmids\": [\"12750295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2 overexpression impairs alpha3beta1 integrin-dependent cell reaggregation and motility on laminin-5; tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin (but not other integrins), and CD81 controls EWI-2 maturation and cell surface localization.\",\n      \"method\": \"Overexpression, immunodepletion, re-expression, cell migration/reaggregation assay, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including immunodepletion, re-expression, and chimeric mutant controls in a well-cited study\",\n      \"pmids\": [\"14662754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EWI-2 directly associates with ezrin-radixin-moesin (ERM) proteins via a basic charged stretch in its cytoplasmic domain, co-localizes with ERMs at microspikes and cellular uropods, and silencing of EWI-2 augments cell migration, polarity, and ERM phosphorylation.\",\n      \"method\": \"GST pulldown, protein-protein binding assay, co-immunoprecipitation, confocal microscopy, dominant-negative moesin, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct in vitro binding assay (GST fusion) combined with dominant-negative experiment and siRNA functional readout, strongly replicated across multiple approaches\",\n      \"pmids\": [\"16690612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2 modulates alpha4beta1 integrin function: wild-type EWI-2 overexpression impairs lymphocyte spreading and ruffling on VCAM-1, reorganizes cell-surface CD81, and increases alpha4beta1-CD81 complex size; a cytoplasmic-tail mutant EWI-2 lacks these effects and fails to associate with alpha4beta1 and CD81.\",\n      \"method\": \"Co-immunoprecipitation, size exclusion chromatography, shear flow adhesion assay, overexpression/mutant analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, mutant controls, and functional assays in the same study\",\n      \"pmids\": [\"15070678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EWI-2/CD316 functions as a receptor for heat shock protein HSPA8 on dendritic cells; soluble EWI-2 binds both HSPA8-expressing cells and immobilized HSPA8 protein, and EWI-2 ligation enhances CCL21-dependent migration of mature dendritic cells while attenuating their antigen-specific stimulatory capacity.\",\n      \"method\": \"Expression cloning, binding assay (soluble EWI-2 to cells/immobilized protein), functional migration and T cell stimulation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — expression cloning identification of ligand, direct binding assay, functional assays in a single study\",\n      \"pmids\": [\"17785435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EWI-2 loss from the oocyte surface in CD9-null mice (reduced to <10% of wild-type levels) depends on CD9 expression, indicating CD9 is required for normal surface localization of EWI-2 on eggs.\",\n      \"method\": \"Genetic knockout (CD9-null mice), flow cytometry/surface protein quantification\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with direct protein quantification, single lab\",\n      \"pmids\": [\"19107828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EWI-2 participates in mouse sperm-egg interaction: anti-IgSF8 antibody has moderate inhibitory effects on sperm-egg binding, while CD9 perturbation significantly inhibits fusion, suggesting IgSF8 and CD9 play discrete roles in gamete interaction.\",\n      \"method\": \"Antibody-mediated perturbation, in vitro fertilization assay\",\n      \"journal\": \"Reproduction, fertility, and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional antibody perturbation assay with direct comparison to CD9, single lab\",\n      \"pmids\": [\"19210920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EWI-2 expression in glioblastoma cells reorganizes tetraspanins CD9 and CD81 (increasing their mutual association) and dissociates CD81 and other tetraspanins from MMP-2 and MT1-MMP, thereby inhibiting invasion and colony formation.\",\n      \"method\": \"Overexpression, co-immunoprecipitation, soft agar and invasion assays, in vivo tumor xenograft\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP showing molecular reorganization combined with in vitro and in vivo functional assays, single lab\",\n      \"pmids\": [\"19107234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A glycine zipper motif in the transmembrane domain of EWI-2 is essential for its interaction with CD81, and palmitoylation on two juxtamembranous cytosolic cysteines is required for EWI-2 interaction with both CD81 and CD9; PIP binding regulates palmitoylation and protein stability but not vice versa.\",\n      \"method\": \"Mutagenesis, palmitoylation assay, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — active-site/domain mutagenesis with direct biochemical readouts for both transmembrane glycine zipper and palmitoylation sites\",\n      \"pmids\": [\"21343309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EWI2 cytoplasmic tail directly binds phosphatidylinositol phosphates (PIPs) via two basic residue clusters; palmitoylation at N-terminal cytoplasmic cysteines is required for tetraspanin-EWI2 association and EWI2-dependent inhibition of cell migration and lamellipodia formation; the two modifications have differential and opposing roles in cell-cell adhesion and proliferation.\",\n      \"method\": \"Lipid-binding assay, site-directed mutagenesis, palmitoylation assay, co-immunoprecipitation, cell migration/lamellipodia assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro lipid-binding assay, mutagenesis of both interaction sites, functional readouts for migration and adhesion\",\n      \"pmids\": [\"21609323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EWI-2 colocalizes with CD3 and CD81 at the central SMAC of the T cell immune synapse; mass spectrometry of EWI-2 cytoplasmic domain pulldowns identified α-actinin as a direct binding partner, regulated by PIP2; silencing of EWI-2 or a cytoplasmic truncation mutant increases IL-2 secretion upon antigen stimulation; EWI-2 and α-actinin-4 silencing each increase HIV cell infectivity.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry pulldown, confocal microscopy, siRNA knockdown, cell-cell fusion assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — MS identification of binding partner confirmed by co-IP, PIP2 regulation shown biochemically, functional assays with siRNA and dominant-negative mutant\",\n      \"pmids\": [\"22689882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (shown by immunoprecipitation) and localizes to puncta within axons and growth cones of olfactory sensory neurons consistent with tetraspanin-enriched microdomain localization; IgSF8 glomerular expression is transitory during synapse formation and is re-induced upon OSN regeneration.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence/confocal microscopy, lesion/regeneration model\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP plus localization during synaptogenesis, single lab, no direct functional manipulation\",\n      \"pmids\": [\"22687584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EWI-2 negatively regulates TGF-β signaling in melanoma by sequestering CD9 and CD81 away from TGF-β receptors; when EWI-2 is knocked down, CD9 and CD81 become available to support TβR2-TβR1 association, markedly elevating TGF-β signaling and downstream EMT, migration, invasion, and metastasis.\",\n      \"method\": \"RNAi, overexpression, co-immunoprecipitation, in vitro cytostasis/migration/invasion assays, in vivo lung metastasis model\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established by showing CD9/CD81 depletion reverses EWI-2 knockdown phenotypes; multiple orthogonal assays in vitro and in vivo\",\n      \"pmids\": [\"25656846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EWI-2 accumulates at the presynaptic terminal of the HIV virological synapse and inhibits Env-mediated cell-cell fusion; EWI-2 is downregulated upon HIV-1 infection (likely by Vpu); EWI-2 and CD81 levels are restored on syncytia surfaces, contributing to prevention of further fusion.\",\n      \"method\": \"Quantitative fluorescence microscopy, shRNA knockdown, cell-cell fusion assay\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by microscopy and functional siRNA knockdown with fusion readout, single lab\",\n      \"pmids\": [\"31757023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IgSF8 is a neuronal receptor enriched in the hippocampal mossy fiber (MF) pathway; presynaptic Igsf8 deletion impairs MF synaptic architecture, robustly decreases bouton filopodia density (providing feedforward inhibition), disrupts excitation/inhibition balance, and increases CA3 pyramidal neuron excitability; cell-surface interactome screening identified IgSF8 binding partners at MF synapses.\",\n      \"method\": \"Cell-surface interactome screening, presynaptic conditional knockout, electron microscopy, electrophysiology, synaptome proteomics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — conditional KO with multiple readouts (ultrastructure, electrophysiology, proteomics), interactome screening\",\n      \"pmids\": [\"33057002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EWI-2 localizes not only on the plasma membrane but also on the nuclear envelope, where it regulates nuclear translocation of EGFR signaling molecules and sorting of miR-3934-5p between cells and exosomes, acting through the EGFR-MAPK-ERK pathway to inhibit prostate cancer cell metastasis.\",\n      \"method\": \"Subcellular fractionation/localization, overexpression/knockdown, miRNA quantification, signaling pathway analysis\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — novel nuclear envelope localization claim with limited mechanistic validation; single lab, partial follow-up\",\n      \"pmids\": [\"33605506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EWI2 prevents EGFR clustering, endocytosis, and activation; upon EWI2 silencing or ablation, EGFR clustering and endocytosis increase, leading to elevated ERK MAP kinase signaling, partial EMT, and increased cancer cell proliferation and metastatic potential; EGFR or ERK inhibition abrogates these phenotypes.\",\n      \"method\": \"siRNA/gene ablation, single-molecule localization microscopy (EGFR clustering), endocytosis assay, signaling pathway inhibition, in vivo xenograft\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — super-resolution imaging of EGFR clustering, rescue by pathway inhibitors, in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"35773608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EWI2 promotes endolysosomal degradation of growth factor receptors and integrins by elevating lysosome formation via nuclear retention of TFEB (the master lysosomogenesis transcription factor), reducing cell-surface levels of these receptors and suppressing lung cancer growth and metastasis.\",\n      \"method\": \"EWI2 forced expression, lysosome assays, receptor surface/total level quantification, TFEB nuclear localization assay, in vivo tumor model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — TFEB nuclear retention mechanistically linked to lysosome formation and receptor degradation with in vivo validation, single lab\",\n      \"pmids\": [\"35339615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IGSF8 expressed on tumor cells suppresses NK cell function by directly interacting with inhibitory NK cell receptors KIR3DL2 (human) and Klra9 (mouse); an antibody blocking this interaction enhances NK cell killing in vitro and upregulates antigen presentation, NK cytotoxicity, and T cell signaling in vivo, inhibiting tumor growth alone or in combination with anti-PD1.\",\n      \"method\": \"CRISPR screen, co-immunoprecipitation/interaction assay, antibody blockade, in vitro NK killing assay, syngeneic tumor models\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — CRISPR screen discovery validated by direct binding, functional in vitro and in vivo assays, antibody rescue experiment with multiple orthogonal readouts\",\n      \"pmids\": [\"38657602\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 (requiring a glycine zipper in its transmembrane domain and palmitoylation of cytoplasmic cysteines), links tetraspanin-enriched microdomains to the actin cytoskeleton via direct binding to ERM proteins and α-actinin (regulated by PIP2 and PIPs), modulates integrin (α3β1, α4β1) and growth factor receptor (EGFR) function by controlling their clustering, endocytosis, and endolysosomal degradation, negatively regulates TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, acts as an innate immune checkpoint by interacting with NK cell inhibitory receptors KIR3DL2/Klra9, organizes hippocampal CA3 microcircuit connectivity as a presynaptic regulator of mossy fiber bouton filopodia and E/I balance, and inhibits HIV-induced cell-cell fusion at the virological presynapse.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that organizes tetraspanin-enriched microdomains and modulates integrin, growth factor receptor, and immune receptor signaling. It forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 — requiring a transmembrane glycine zipper motif and palmitoylation of cytoplasmic cysteines — and links these microdomains to the actin cytoskeleton through direct binding of ERM proteins and α-actinin, regulated by phosphoinositide lipids [PMID:11504738, PMID:21343309, PMID:16690612, PMID:22689882]. IGSF8 negatively regulates cell migration, EGFR clustering/endocytosis, and TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors and by promoting endolysosomal degradation of growth factor receptors and integrins via TFEB-dependent lysosomogenesis, thereby suppressing EMT and metastasis [PMID:25656846, PMID:35773608, PMID:35339615]. IGSF8 also functions as an innate immune checkpoint that suppresses NK cell cytotoxicity through direct interaction with inhibitory receptors KIR3DL2 and Klra9, and serves as a presynaptic regulator in hippocampal mossy fiber circuits controlling bouton filopodia density and excitation/inhibition balance [PMID:38657602, PMID:33057002].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of IGSF8 as a direct, highly stoichiometric partner of tetraspanins CD9 and CD81 established it as a core component of tetraspanin-enriched microdomains, resolving which IgSF proteins are bona fide tetraspanin partners versus fortuitous co-precipitants.\",\n      \"evidence\": \"Co-purification, mass spectrometry, chemical cross-linking, and immunodepletion in multiple cell lines; domain-deletion mapping of the Ig3–Ig4 domains required for CD81 binding\",\n      \"pmids\": [\"11504738\", \"11673522\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of the IGSF8–tetraspanin interface not resolved at atomic level\",\n        \"Whether IGSF8 can engage CD9 and CD81 simultaneously or competitively was not determined\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"IGSF8 was shown to modulate integrin-dependent adhesion and migration by reorganizing tetraspanin–integrin complexes, revealing a mechanism by which IgSF8 indirectly controls integrin function through tetraspanin scaffolding rather than direct integrin binding.\",\n      \"evidence\": \"Overexpression and cytoplasmic-tail mutant analysis in cell reaggregation, migration, and shear-flow adhesion assays; co-IP with α3β1 and α4β1 integrins; chimeric CD9/CD82 domain mapping of the EWI-2 binding regions on CD9\",\n      \"pmids\": [\"14662754\", \"15070678\", \"12708969\", \"12750295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether IGSF8 controls integrin signaling through conformational or purely spatial mechanisms was not distinguished\",\n        \"Endogenous stoichiometry of IGSF8–tetraspanin–integrin complexes in primary cells not established\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that IGSF8 directly binds ERM proteins via a basic cytoplasmic stretch and that its silencing augments ERM phosphorylation and cell migration established IGSF8 as a negative regulator linking tetraspanin microdomains to the cortical actin cytoskeleton.\",\n      \"evidence\": \"GST pulldown (direct binding), confocal co-localization at microspikes/uropods, dominant-negative moesin, siRNA knockdown\",\n      \"pmids\": [\"16690612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ERM binding and tetraspanin binding are mutually exclusive or cooperative was not resolved\",\n        \"Upstream signals controlling IGSF8–ERM dissociation remain undefined\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mutagenesis of the transmembrane glycine zipper and cytoplasmic palmitoylation sites, together with phosphoinositide-binding mapping, revealed the post-translational logic governing IGSF8 assembly into tetraspanin webs and its regulation of migration and adhesion.\",\n      \"evidence\": \"Site-directed mutagenesis of TM glycine zipper and palmitoylation cysteines, in vitro lipid-binding assays, palmitoylation assays, functional migration/lamellipodia assays\",\n      \"pmids\": [\"21343309\", \"21609323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the palmitoyl acyltransferase(s) modifying IGSF8 unknown\",\n        \"How PIP binding and palmitoylation are dynamically coordinated in live cells not shown\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identification of α-actinin as a PIP2-regulated direct cytoplasmic partner of IGSF8, and the finding that IGSF8 localizes to the immune synapse cSMAC where its loss elevates IL-2 secretion and HIV infectivity, established IGSF8 as a functional brake on T cell activation and viral entry.\",\n      \"evidence\": \"Mass spectrometry pulldown, co-IP, PIP2 regulation, confocal at immune synapse, siRNA/cytoplasmic-truncation functional assays, cell–cell fusion assay\",\n      \"pmids\": [\"22689882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether α-actinin and ERM binding are competitive or spatially segregated was not tested\",\n        \"Mechanism by which IGSF8 restrains IL-2 signaling beyond clustering is undefined\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Epistasis experiments demonstrated that IGSF8 negatively regulates TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, so that IGSF8 loss frees tetraspanins to promote TβR2–TβR1 association, driving EMT and metastasis — a new signaling-level mechanism for IGSF8's tumor-suppressive activity.\",\n      \"evidence\": \"RNAi/overexpression epistasis (CD9/CD81 depletion rescues EWI-2 knockdown phenotype), co-IP of TGF-β receptor complexes, in vivo lung metastasis model\",\n      \"pmids\": [\"25656846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether IGSF8 physically contacts TGF-β receptors or acts exclusively through tetraspanin redistribution not clarified\",\n        \"Applicability beyond melanoma not tested\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Conditional presynaptic knockout of Igsf8 in hippocampal mossy fibers revealed a non-immune, neurodevelopmental role: IGSF8 is required for bouton filopodia formation and excitation/inhibition balance in CA3 circuits, establishing it as a synaptic organizer.\",\n      \"evidence\": \"Presynaptic conditional knockout, electron microscopy, electrophysiology, cell-surface interactome screening\",\n      \"pmids\": [\"33057002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Trans-synaptic binding partners mediating filopodia formation not fully identified\",\n        \"Whether IGSF8 synaptic function depends on tetraspanin association was not tested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Super-resolution imaging and gene ablation showed that IGSF8 prevents EGFR clustering and endocytosis, and that IGSF8 promotes endolysosomal degradation of growth factor receptors and integrins via TFEB-dependent lysosomogenesis, providing two complementary mechanisms for receptor downregulation and tumor suppression.\",\n      \"evidence\": \"Single-molecule localization microscopy (EGFR clustering), endocytosis assays, TFEB nuclear localization assay, EGFR/ERK inhibitor rescue, in vivo xenograft and lung cancer models\",\n      \"pmids\": [\"35773608\", \"35339615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How IGSF8 promotes TFEB nuclear retention mechanistically is unclear\",\n        \"Whether EGFR clustering prevention and lysosome-mediated degradation operate in the same or distinct cell contexts is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CRISPR screening and direct binding studies revealed IGSF8 as an innate immune checkpoint: tumor-expressed IGSF8 engages inhibitory NK receptors KIR3DL2/Klra9 to suppress NK cell killing, and antibody blockade of this axis enhances antitumor immunity alone or with anti-PD1.\",\n      \"evidence\": \"CRISPR screen, co-immunoprecipitation/interaction assay, antibody blockade, in vitro NK killing assay, syngeneic tumor models\",\n      \"pmids\": [\"38657602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of IGSF8–KIR3DL2 interaction not determined\",\n        \"Whether tetraspanin association modulates the immune checkpoint function is unknown\",\n        \"Patient-level correlation between IGSF8 expression and NK cell infiltration/response to immunotherapy not yet established\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of IGSF8's multivalent interactions (tetraspanins, KIR3DL2, ERM, α-actinin), whether its immune checkpoint and tetraspanin-scaffolding functions are mechanistically coupled, and the identity of its trans-synaptic partners in mossy fiber circuits.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No atomic-resolution structure of IGSF8 or its complexes available\",\n        \"Functional interdependence of immune checkpoint and tetraspanin-organizing roles untested\",\n        \"Trans-synaptic ligands at mossy fiber synapses remain unidentified\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 6, 15, 19, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [5, 13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 11, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 5, 8, 13, 16, 17, 21]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [15, 19, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 21]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [4, 6, 9]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [10, 15, 19, 20]}\n    ],\n    \"complexes\": [\n      \"Tetraspanin-enriched microdomain (CD9/CD81-EWI-2)\"\n    ],\n    \"partners\": [\n      \"CD9\",\n      \"CD81\",\n      \"EZR\",\n      \"ACTN4\",\n      \"KIR3DL2\",\n      \"CD82\",\n      \"EGFR\",\n      \"HSPA8\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}